本文選題：玉米 + 凍害��； 參考：《東北農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：隨著溫室氣體排放增加,全球氣候變暖導(dǎo)致極端天氣事件發(fā)生頻率明顯增加,各種極端氣候事件,如強(qiáng)寒潮、干旱和洪澇災(zāi)害等對(duì)農(nóng)業(yè)生產(chǎn)造成了嚴(yán)重影響。玉米是世界上最重要的糧食作物、飼用作物和能源作物之一。而且由于起源于熱帶地區(qū),玉米是典型喜溫喜光的C4植物,對(duì)溫度條件要求較高,易受低溫冷(凍)害的影響。在我國(guó)玉米生產(chǎn)中,春季和秋季的霜凍對(duì)玉米產(chǎn)量易造成嚴(yán)重影響,尤其是對(duì)北方春播玉米區(qū)和西北灌溉玉米區(qū),而且春季霜凍的頻次要高于秋季霜凍。東北地區(qū)是我國(guó)玉米的主產(chǎn)區(qū),而黑龍江省玉米常年種植面積已達(dá)到糧食作物總面積的一半以上,為我國(guó)玉米播種面積最大的省份。近年來由于玉米需求的剛性增長(zhǎng)和玉米種植比較效益的提高,黑龍江省玉米種植區(qū)域已經(jīng)發(fā)展到第6積溫帶,玉米跨區(qū)種植的情況非常嚴(yán)重。與此同時(shí),近年來黑龍江省春季常有雨雪冰凍天氣,玉米播種期易遇倒春寒而推遲,直接影響玉米的正常播種,而且玉米幼苗期也易遭受倒春寒而出現(xiàn)凍害損傷,影響玉米的正常生長(zhǎng),并最終造成減產(chǎn)。因此,開展玉米苗期抗凍性研究,篩選玉米抗凍種質(zhì)資源,探究玉米苗期抗凍性生理生化機(jī)制,挖掘玉米在低溫脅迫下的抗凍基因,進(jìn)而開展玉米抗凍性分子標(biāo)記輔助育種等相關(guān)方面的研究,對(duì)于解決黑龍江省玉米冷(凍)害問題具有重要而深遠(yuǎn)的理論和實(shí)踐意義。本研究利用30份黑龍江省常用玉米自交系,開展了玉米苗期抗凍性鑒定及相關(guān)生理指標(biāo)測(cè)定,同時(shí)結(jié)合轉(zhuǎn)錄組測(cè)序技術(shù),對(duì)具有不同抗凍性的玉米自交系苗期凍害脅迫響應(yīng)基因及相關(guān)代謝通路進(jìn)行了比較分析,研究結(jié)果如下:一、玉米苗期抗凍性的高效鑒定方法本研究選取三葉一心期的玉米自交系幼苗放置于-1℃的培養(yǎng)箱中分別進(jìn)行1h、3h和5h的凍害處理,之后放置于正常培養(yǎng)條件的培養(yǎng)箱中恢復(fù)3天,3天之后統(tǒng)計(jì)幼苗存活率,以幼苗存活率來作為抗凍性的測(cè)量指標(biāo)。結(jié)果顯示凍害脅迫處理3小時(shí)的幼苗存活率從5.01%到97.3%不等,呈現(xiàn)正態(tài)分布,能夠有效的區(qū)分不同自交系的抗凍性,因此將凍害脅迫時(shí)間3小時(shí)作為有效鑒定玉米苗期自交系抗凍性的處理時(shí)間。并且篩選出兩份極端自交系用于后續(xù)研究,其中,自交系KR701在凍害處理3小時(shí)之后的幼苗存活率為97.3%,作為抗凍自交系;Hei8834在凍害處理3小時(shí)之后的幼苗存活率為5.01%,作為凍害敏感自交系。二、凍害脅迫下玉米苗期生理機(jī)制分析Hei8834在凍害處理之后表現(xiàn)出明顯的葉片萎蔫、失綠,臺(tái)盼藍(lán)染色后顯微觀察也發(fā)現(xiàn)凍害處理之后Hei8834的葉片氣孔張開并呈現(xiàn)深藍(lán)色,說明其葉片受凍害損傷嚴(yán)重。同時(shí),凍害處理后,Hei8834的相對(duì)電導(dǎo)率在0.5小時(shí)和1小時(shí)分別為0.13和0.87,表明Hei8834在凍害處理0.5小時(shí)到1小時(shí)之間凍害損傷明顯加劇;同時(shí)超氧化物歧化酶活性在凍害處理后1小時(shí)達(dá)到最高值57.3 U/mg,之后便開始下降;過氧化物酶活性在凍害處理后0.5小時(shí)達(dá)到最大值53.7 U/mg,之后也呈現(xiàn)下降趨勢(shì);可溶性蛋白和可溶性糖含量都是在凍害處理之后一直緩慢升高。KR701的相對(duì)電導(dǎo)率在凍害處理之后一直維持0.2以下;超氧化物歧化酶活性在凍害處理之后上升趨勢(shì)高于Hei8834,而且在2小時(shí)達(dá)到最高值80.8,之后略有降低;過氧化物酶活性在凍害處理之后一直處于上升趨勢(shì);可溶性蛋白含量在凍害處理之后也是上升趨勢(shì);可溶性糖含量在凍害處理之后也是一直升高,直到2小時(shí)之后維持相對(duì)穩(wěn)定水平,并有下降趨勢(shì)。因此,凍害敏感自交系Hei8834在凍害處理1小時(shí)之后,凍害損傷明顯,凍害抵抗機(jī)制已失效;而抗凍自交系KR701在凍害處理3小時(shí)以內(nèi),凍害損傷較小,凍害抵抗機(jī)制有效的保護(hù)了細(xì)胞免于凍害損傷,但是在2小時(shí)到3小時(shí)之間,有些生理指標(biāo)(超氧化物歧化酶活性和可溶性糖)已經(jīng)開始略微呈現(xiàn)下降趨勢(shì),暗示凍害處理時(shí)間如再延長(zhǎng),抗凍自交系KR701的抗凍機(jī)制也不能有效抵抗凍害脅迫。三、不同抗凍性玉米自交系幼苗凍害脅迫前后的轉(zhuǎn)錄組測(cè)序分析對(duì)這兩份極端材料在幼苗期凍害處理之前和凍害處理之后進(jìn)行轉(zhuǎn)錄組測(cè)序,共得到大約154.4百萬長(zhǎng)度為101 bp的序列,其中78.84%(121.8百萬)的序列能比對(duì)在玉米參考基因組Zea_mays_Ensembl_AGPv3。聚類分析及主成分分析(PCA)結(jié)果顯示,抗凍自交系KR701在凍害處理之前和凍害處理之后的樣本歸為一類,凍害敏感自交系Hei8834在凍害處理之前和凍害處理之后的樣本歸為一類,導(dǎo)致不同樣本間基因表達(dá)量最大差別的因素為自交系之間的差異。4個(gè)不同處理的7個(gè)樣本中,共有19794個(gè)基因表達(dá),其中有360個(gè)基因在抗凍自交系KR701中經(jīng)過凍害脅迫之后特異表達(dá),489個(gè)基因在凍害敏感自交系(Hei8834)在凍害脅迫之后特異表達(dá)。共有4550個(gè)基因在4個(gè)不同的處理(兩份自交系之間以及凍害處理前后)中的表達(dá)量存在差異(即為差異表達(dá)基因,DEG)。對(duì)這些特異表達(dá)基因和差異表達(dá)基因分別進(jìn)行了基因本體論分析(GO分析),結(jié)果發(fā)現(xiàn)凍害處理后凍害敏感自交系特異表達(dá)的基因與抗凍自交系特異表達(dá)的基因相比,特異的參與了一些生物學(xué)過程,如低溫響應(yīng)、有機(jī)酸轉(zhuǎn)運(yùn),此外泛素連接酶復(fù)合體組分也顯著富集。這也從轉(zhuǎn)錄水平上說明凍害敏感自交系在凍害處理中受到的損傷較為嚴(yán)重,從而觸發(fā)了更多的生物學(xué)過程。同時(shí),雖然許多GO條目在兩個(gè)自交系凍害處理后特異表達(dá)的基因中都有出現(xiàn),但是這些GO條目在兩個(gè)自交系所富集的顯著程度不同,說明參與同一GO的基因在兩個(gè)自交系中所占的比例是不同的。對(duì)抗凍自交系凍害處理前后的439個(gè)差異基因和凍害敏感自交系凍害處理前后的852個(gè)差異基因分別進(jìn)行了GO分析,結(jié)果發(fā)現(xiàn)在生物學(xué)過程分類中,抗凍自交系中富集最為顯著的是凍害響應(yīng),而凍害敏感自交系富集最為顯著的是低溫響應(yīng);在分子功能分類中,抗凍自交系中富集最為顯著的是結(jié)合活性,而凍害敏感自交系富集最為顯著的是蛋白質(zhì)酪氨酸激酶活性;在細(xì)胞組分分類中,抗凍自交系中有三個(gè)GO條目顯著富集,均為膜組分相關(guān),而凍害敏感自交系富集最為顯著的是泛素連接酶復(fù)合體。此外我們對(duì)在兩個(gè)自交系間凍害響應(yīng)存在顯著差異的基因進(jìn)行了擬南芥同源基因功能檢索,結(jié)果鑒定到許多參與逆境響應(yīng)和植物激素信號(hào)通路相關(guān)的基因。這些基因的后續(xù)分析將會(huì)增加我們對(duì)玉米幼苗在早期凍害脅迫下響應(yīng)凍害的分子機(jī)制的認(rèn)識(shí),并對(duì)玉米抗低溫育種提供良好的育種策略。最后,挑選出30個(gè)基因進(jìn)行實(shí)時(shí)熒光定量PCR(qRT-PCR)驗(yàn)證,結(jié)果顯示轉(zhuǎn)錄組測(cè)序結(jié)果和qRT-PCR驗(yàn)證結(jié)果在抗凍材料和凍害敏感材料中的相關(guān)系數(shù)分別是80%和50%,說明轉(zhuǎn)錄組分析結(jié)果與qRT-PCR結(jié)果吻合度較高。
[Abstract]:With the increase of greenhouse gas emissions, global warming has led to an obvious increase in the frequency of extreme weather events, and a variety of extreme weather events, such as strong cold tide, drought and flood, have seriously affected agricultural production. Corn is one of the most important food crops, forage crops and energy crops in the world. In the region, maize is a typical warm and warm C4 plant with high temperature and cold (freezing) damage. In maize production in China, spring and autumn frost have a serious effect on maize yield, especially in northern spring sowing corn and northwest irrigated corn region, and the frequency of frost in spring is higher than that in autumn frost. The northeast region is the main producing area of Maize in China, and the annual planting area of Maize in Heilongjiang has reached more than half of the total area of grain crops, which is the largest province in China. In recent years, because of the rigid growth of corn demand and the improvement of the comparative benefit of Maize planting in recent years, the maize planting area in Heilongjiang has developed to the first. In the 6 product of temperate zone, the situation of Maize Cross planting is very serious. At the same time, in recent years, the spring of Heilongjiang often has the freezing weather of rain and snow, the corn sowing period is easy to meet the spring cold and postpone, directly affects the normal sowing of corn, and the maize seedling stage is also vulnerable to the cold injury, which affects the normal growth of corn, and eventually causes the corn. Therefore, to carry out the research on the frost resistance of Maize at the seedling stage, select the germplasm resources of the corn frost resistance, explore the physiological and biochemical mechanism of the cold resistance of Maize at the seedling stage, excavate the antifreeze gene of corn under the low temperature stress, and then carry out the research on the related aspects of the corn frost resistance molecular marker assisted breeding, which can solve the problem of cold (freezing) damage to Maize in Heilongjiang province. There are important and far-reaching theoretical and practical significance. In this study, 30 common maize inbred lines in Heilongjiang province were used to identify the resistance of Maize at seedling stage and to determine the related physiological indexes. At the same time, the response genes and related metabolic pathways of the cold damage stress of maize inbred lines with different frost resistance were carried out in combination with the sequencing of the transcriptional sequence. The results are as follows: 1. High efficiency identification method for the frost resistance of Maize at the seedling stage. This study selected the maize inbred lines of the three leaves and one heart stage to carry out the freezing injury treatment of 1H, 3H and 5h respectively in the -1 C incubator. Then the seedlings were placed in the normal culture incubator for 3 days, and the seedling survival rate was counted after 3 days, and the seedlings were counted. The survival rate was used as an indicator of frost resistance. The results showed that the survival rate of the seedlings for 3 hours was from 5.01% to 97.3%, showing a normal distribution, which could effectively distinguish the frost resistance of different inbred lines. So 3 hours of freezing stress time was used as an effective method to identify the freezing resistance of maize inbred lines. Two extreme self inbred lines were used for follow-up studies. Among them, the survival rate of the seedlings of the inbred line KR701 after 3 hours of freezing injury was 97.3%, as a freeze-resistant inbred line. The survival rate of the seedlings after 3 hours of freezing injury treatment was 5.01%, as a freezing sensitive inbred line. Two, the physiological mechanism of Maize Seedling under the frost stress was analyzed by Hei8834 in the frost injury. After the treatment, the leaf blowhole of Hei8834 was open and dark blue after the freezing injury treatment, indicating that the blade was damaged by frost damage. At the same time, the relative conductivity of Hei8834 was 0.13 and 0.87, respectively, when the freezing injury treatment was 0.5 and 1 hours, indicating that Hei8834 was frozen. At the same time, the activity of superoxide dismutase (SOD) reached a maximum value of 57.3 U/mg at 1 hours after the treatment of freezing injury, and then began to decrease, and the peroxidase activity reached the maximum value of 53.7 U/mg at 0.5 hours after the freezing injury treatment, and then it also showed a downward trend, and soluble protein and soluble sugar contained the soluble sugar. The relative conductivity of.KR701 has been increased slowly after the freezing injury treatment, and the relative conductivity of the.KR701 has been maintained below 0.2 after the freezing injury treatment, and the activity of superoxide dismutase is higher than that of Hei8834 after freezing injury treatment, and it reaches the maximum value of 80.8 at 2 hours, then decreases slightly, and the activity of peroxidase is always in the cold injury treatment. The content of soluble protein was also rising after freezing injury treatment, and the content of soluble sugar increased after freezing injury treatment, and maintained relative stability level until 2 hours after 2 hours. Therefore, the frost damage sensitive self inbred line was obviously damaged after 1 hours of freezing injury treatment, and the mechanism of freezing injury resistance was already found. While the freezing damage treatment was less than 3 hours, the frost damage was less than 3 hours, and the freezing injury resistance mechanism effectively protected the cell from freezing damage, but between 2 hours and 3 hours, some physiological indexes (superoxide dismutase activity and soluble sugar) had begun to decrease slightly, suggesting the freezing injury treatment time, such as The frost resistance mechanism of the anti freeze inbred line KR701 could not effectively resist the frost damage. Three, the transcriptional sequence analysis of the different frost resistance maize inbred lines before and after the freezing stress of the two extreme materials was sequenced before the freezing injury treatment and the freeze injury of the seedling stage, and a total of about 154 million 400 thousand of the length of 101 BP was obtained. The sequence of 78.84% (121 million 800 thousand) was compared with the results of Zea_mays_Ensembl_AGPv3. cluster analysis and principal component analysis (PCA) in maize reference genome. The results showed that the samples of the antifreeze KR701 inbred line before the freezing injury treatment and the freezing injury treatment were classified as a class. The freezing injury sensitized inbred line Hei8834 was before the freezing injury treatment and the freezing injury treatment. The following samples are classified into one class. The factors that lead to the maximum difference in gene expression among different samples are 7 different.4 samples of the inbred lines. There are 19794 genes, of which 360 genes are specifically expressed in the anti freeze inbred line KR701 after freezing stress, and the 489 genes are in the cold sensitive inbred line (Hei8834). The expression of 4550 genes in 4 different treatments (between two inbred lines and before and after freezing injury treatment) were different (i.e., differentially expressed genes, DEG). The gene ontology analysis (GO analysis) of these specific and differentially expressed genes was carried out, and the results of freezing injury treatment were found. The specific genes expressed in the sensitive inbred lines of the post freeze injury are specifically involved in some biological processes, such as the low temperature response, the organic acid transport, and the ubiquitin ligase complex components, as well as the damage caused by the freezing injury sensitive inbred lines in the freezing injury treatment. More serious and more biological processes were triggered. While many of the GO entries appeared in the two inbred line specific genes after freezing injury treatment, these GO entries were significantly different in the two inbred lines, indicating that the proportion of the genes involved in the same GO is different in the two inbred lines. 852 different genes before and after freezing injury treatment before and after freezing injury treatment of frozen self inbred lines were analyzed by GO. The results showed that in the biological process classification, the most significant accumulation of freezing injury in the antifreeze inbred lines was the freezing injury response, and the most significant accumulation of cold damage sensitive inbred lines was the low temperature response. In the molecular functional classification, the most significant accumulation of the antifreeze inbred lines is the binding activity, while the most significant enrichment of the freezing sensitive inbred lines is the protein tyrosine kinase activity. In the cell classification, three GO entries are significantly enriched in the antifreeze inbred lines, all of which are related to the membrane components, and the most significant enrichment of the freezing sensitive inbred lines is the most significant. It is the ubiquitin ligase complex. In addition, we searched the Arabidopsis homologous gene for genes of Arabidopsis homologous genes with significant differences in the freezing damage response between two inbred lines. The results identified many genes involved in stress response and plant hormone signaling pathways. Subsequent analysis of these genes will increase our early growth in maize seedlings. A good breeding strategy was provided for maize breeding in response to cold damage under the cold damage stress. Finally, 30 genes were selected for real-time fluorescence quantitative PCR (qRT-PCR) verification. The results showed that the correlation coefficient of the transcriptional sequence and qRT-PCR verification results in the antifreeze material and the freezing sensitive material was 8, respectively. 0% and 50%, indicating that the transcriptome analysis results are in good agreement with the qRT-PCR results.

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S513;S42

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